Mold was nominated to the NTP for toxicological characterization and based on their inhalation, mycological, and immunological expertise, NIOSH was engaged as a partner to conduct multiple sub-chronic (13-week) exposure studies to individual organisms or mixtures of molds mimicking a real-world exposure. NIOSH has developed a system that allows for precisely controlled exposures to particulates via inhalation. This approach overcomes a number of limitations associated with previous mold exposure studies in experimental animal models that have used less relevant means of exposure to assess fungal toxicity. The acoustical generation system (AGS) developed as part of this partnership delivers fungal test articles to mice in a manner that simulates natural indoor fungal contaminant exposure. NIOSH continues to use the AGS and state-of-the-art methodologies to characterize toxicological endpoints following subchronic dry fungal spore and particle exposures. Following the completion of an NTP Study to evaluate the effects of exposure to Aspergillus fumigatus, NIOSH has begun studies to characterize pulmonary immune responses to Stachybotrys chartarum (mycotoxin producing chemotype). A S. chartarum dosimetry study identified doses that resulted in an immunologic response but not overt toxicity. Preliminary data derived from 4 week and 13 week exposures showed an early Th2 response after 4 weeks of exposure that shifted toward a Th1 dominant response after 13 weeks of subchronic inhalation exposure to S. chartarum conidia. Macrophages were elevated after 4 and 13 weeks of exposure, along with increased arg1, retnla and il33 expression, that suggested ongoing inflammation. Eosinophil levels were dramatically increased after 4 weeks but were decreasing by 13 weeks, potentially attributed to increased ccl11 and cxcl5. A target organ toxicology study that evaluated a mycotoxin producing chemotype of S. chartarum, was also completed this year and histopathology assessment of the tissues is underway. In addition to the evaluation of the high mycotoxin producing S. chartarum strain, NIOSH developed a method to heat inactivate S. chartarum spores and optimized the growth conditions of a low mycotoxin producing strain of S. chartarum. A 13 week pulmonary immunology study of this strain of S. chartarum is ongoing.